JPWO2014051111A1 - Hepatitis C virus particle formation promoter and method for producing hepatitis C virus particles - Google Patents
Hepatitis C virus particle formation promoter and method for producing hepatitis C virus particles Download PDFInfo
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- JPWO2014051111A1 JPWO2014051111A1 JP2014538656A JP2014538656A JPWO2014051111A1 JP WO2014051111 A1 JPWO2014051111 A1 JP WO2014051111A1 JP 2014538656 A JP2014538656 A JP 2014538656A JP 2014538656 A JP2014538656 A JP 2014538656A JP WO2014051111 A1 JPWO2014051111 A1 JP WO2014051111A1
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Abstract
培養細胞においてHCV粒子の形成を促進しうるHCV粒子形成促進剤を提供し、更にはHCV粒子の産生増強方法を提供する。また、抗HCV剤候補物質の評価方法、並びにHCVワクチンの製造方法を提供する。スタチン又はその薬学的に許容しうる塩を有効成分とするHCV粒子形成促進剤による。スタチン又はその薬学的に許容しうる塩をHCV感染培養細胞に加えることで、感染性HCV粒子が形成促進され、産生増強が認められる。また、当該HCV粒子形成促進剤と抗HCV剤候補物質の存在下にHCV感染細胞を培養することにより抗HCV剤候補物質を評価する。更に、HCV粒子の産生増強方法により産生されたHCV粒子を用いてHCVワクチンを作製する。The present invention provides an HCV particle formation promoter that can promote the formation of HCV particles in cultured cells, and further provides a method for enhancing production of HCV particles. Moreover, the evaluation method of an anti-HCV agent candidate substance and the manufacturing method of HCV vaccine are provided. According to an HCV particle formation promoter containing a statin or a pharmaceutically acceptable salt thereof as an active ingredient. By adding a statin or a pharmaceutically acceptable salt thereof to HCV-infected cultured cells, formation of infectious HCV particles is promoted, and production enhancement is observed. In addition, the anti-HCV agent candidate substance is evaluated by culturing HCV-infected cells in the presence of the HCV particle formation promoter and the anti-HCV agent candidate substance. Furthermore, an HCV vaccine is produced using HCV particles produced by the method for enhancing production of HCV particles.
Description
本発明は、C型肝炎ウイルス(HCV:Hepatitis C Virus)感染培養細胞におけるHCV粒子の形成促進剤及びHCV粒子の産生増強方法に関する。 The present invention relates to an agent for promoting formation of HCV particles and a method for enhancing production of HCV particles in cultured cells infected with hepatitis C virus (HCV).
本出願は、参照によりここに援用されるところの日本出願、特願2012−215474号優先権を請求する。 This application claims the Japanese application and Japanese Patent Application No. 2012-215474 priority which are used here by reference.
1989年にHCV遺伝子がクローニングされてから、C型肝炎患者血清中のHCVを培養細胞に感染させウイルス増殖細胞系を作製する方法の確立はHCV研究の最大のテーマであった。 Since the HCV gene was cloned in 1989, the establishment of a method for infecting cultured cells with HCV in the serum of patients with hepatitis C and creating a virus-proliferating cell line has been the biggest theme of HCV research.
全世界で2億人、日本国内でも約200万人ものHCVキャリヤーが存在している。HCVの感染者は非常に高い確率で慢性肝炎を発症し、一部の患者においては慢性肝炎が更に肝硬変、肝癌へと進行する。つまりHCVは重篤な疾患を引き起こす原因ウイルスである。現在、多くのC型肝炎患者に対してインターフェロン(IFN)、及び抗ウイルス薬であるリバビリンを併用した治療方法が多用されている。しかしながら、IFNが効きにくいタイプのHCV感染者も多く、上記治療方法では治療効果が低いという問題点がある。またIFNは、患者に対して強い副作用を示すことも明らかになっている。そのため、HCVの感染拡大の防止及びHCVの撲滅に向けた、新規治療薬及び新規ワクチンの開発が急務となっている。 There are 200 million HCV carriers worldwide, and about 2 million HCV carriers in Japan. Those infected with HCV develop chronic hepatitis with a very high probability, and in some patients, chronic hepatitis further progresses to cirrhosis and liver cancer. In other words, HCV is a causative virus that causes serious diseases. Currently, treatment methods using interferon (IFN) and ribavirin, an antiviral drug, are frequently used for many patients with hepatitis C. However, there are many types of HCV infected people who are difficult to work with IFN, and there is a problem that the therapeutic effect is low with the above-mentioned treatment method. IFN has also been shown to have strong side effects on patients. Therefore, there is an urgent need to develop new therapeutic drugs and new vaccines to prevent the spread of HCV infection and to eradicate HCV.
一方、HCVの感染性クローンは、動物実験レベルではチンパンジーやヒト肝細胞を移植されたキメラマウスでのみ増殖が可能だが、患者検体から培養細胞に容易に感染できるウイルスは得られておらず、倫理的な問題やコストの面からチンパンジーの利用は進まず、研究面での貢献は厳しかった。HCVのサブゲノムを培養細胞中にて複製増殖させることが可能なHCVサブゲノムRNAレプリコンシステムが作製されたことが報告された(非特許文献1〜4、特許文献1、2)。これにより、培養細胞を用いてHCVの複製機構を解析することが可能となった。これらのHCVサブゲノムRNAレプリコンは、HCVゲノムRNAの5'非翻訳領域中のHCV IRESの下流に存在する構造タンパク質をコードする領域を、ネオマイシン耐性遺伝子及びその下流に連結したEMCV-IRESによって置換したものである。このRNAレプリコンを、ヒト肝癌細胞Huh7に導入してネオマイシン存在下で培養することにより、Huh7細胞内でRNAレプリコンが自律複製することが証明された。 On the other hand, infectious clones of HCV can be propagated only in chimeric mice transplanted with chimpanzees and human hepatocytes at the animal experimental level, but no virus that can easily infect cultured cells from patient specimens has been obtained. The use of chimpanzees did not progress due to problems and costs, and the contribution in research was severe. It was reported that the HCV subgenomic RNA replicon system capable of replicating and proliferating the HCV subgenome in cultured cells was produced (Non-patent Documents 1 to 4, Patent Documents 1 and 2). As a result, it became possible to analyze the replication mechanism of HCV using cultured cells. In these HCV subgenomic RNA replicons, the region encoding the structural protein present downstream of HCV IRES in the 5 'untranslated region of HCV genomic RNA is replaced by the neomycin resistance gene and EMCV-IRES linked downstream thereof. It is. By introducing this RNA replicon into human hepatoma cell Huh7 and culturing it in the presence of neomycin, it was proved that the RNA replicon replicates autonomously in Huh7 cells.
この後、HCVの全ゲノムRNAが自律複製するHCVフルゲノムRNAレプリコンが作製され、さらにHCVの生活環(ウイルス吸着・侵入からウイルス粒子の形成と細胞外への放出まで)を反映する感染増殖系が作製され、報告されている(非特許文献5〜7)。ワクチン等をより効果的に開発するためには、さらに効果的に、感染細胞内でのHCV粒子の形成と細胞外への放出が行われる系が望まれる。 After this, an HCV full-genome RNA replicon that autonomously replicates the entire HCV genomic RNA was created, and an infectious propagation system that reflected the life cycle of HCV (from virus adsorption / invasion to virus particle formation and extracellular release) It has been produced and reported (Non-Patent Documents 5 to 7). In order to develop vaccines and the like more effectively, a system that can more effectively form HCV particles in infected cells and release them outside the cell is desired.
本発明は、培養細胞においてHCV粒子の形成を促進しうるHCV粒子形成促進剤を提供することを課題とし、更にはHCV粒子の産生増強方法を提供することを課題とする。また、抗HCV剤候補物質の評価方法、並びにHCVワクチンの作製方法を提供することを課題とする。 An object of the present invention is to provide an HCV particle formation promoter that can promote the formation of HCV particles in cultured cells, and further to provide a method for enhancing production of HCV particles. It is another object of the present invention to provide a method for evaluating an anti-HCV drug candidate substance and a method for producing an HCV vaccine.
本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、スタチン又はその薬学的に許容しうる塩が効果的にHCV粒子の形成を促進することを見出し、本発明のHCV粒子形成促進剤に係る発明を完成した。また、当該当該HCV粒子形成促進剤と抗HCV剤候補物質の存在下にHCV感染細胞を培養することで抗HCV剤候補物質を評価しうることを見出し、抗HCV剤候補物質の評価方法に係る本発明も完成した。更にHCV粒子産生増強方法により産生されたHCV粒子を用いることでHCVワクチンを作製しうることを見出し、HCVワクチンの作製方法に係る本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors have found that statins or pharmaceutically acceptable salts thereof effectively promote the formation of HCV particles, and the HCV particles of the present invention The invention relating to the formation accelerator was completed. In addition, the present inventors have found that anti-HCV drug candidate substances can be evaluated by culturing HCV-infected cells in the presence of the HCV particle formation promoter and the anti-HCV drug candidate substance, and relate to a method for evaluating anti-HCV drug candidate substances. The present invention has also been completed. Furthermore, it discovered that HCV vaccine could be produced by using the HCV particle produced by the HCV particle production enhancing method, and completed the present invention relating to the method for producing HCV vaccine.
すなわち本発明は、以下よりなる。
1.スタチン又はその薬学的に許容しうる塩を有効成分として含有する、HCV粒子形成促進剤。
2.前記スタチンが、ロバスタチン、フルバスタチン、シンバスタチン、アトルバスタチン及びプラバスタチンからなる群から選択される1又は複数のスタチンである、前項1に記載のHCV粒子形成促進剤。
3.スタチン又はその薬学的に許容しうる塩を産生しうる微生物の培養抽出物を含有する、前項1又は2に記載のHCV粒子形成促進剤。
4.前記微生物がAspergillus属糸状菌である、前項3に記載のHCV粒子形成促進剤。
5.前項1〜4のいずれか1項に記載のHCV粒子形成促進剤の存在下でHCV感染細胞を培養する、HCV粒子の産生増強方法。
6.前項1〜4のいずれか1項に記載のHCV粒子形成促進剤を、前記HCV感染細胞内にHCVタンパク質が形成された後に添加して培養する、前項5に記載のHCV粒子の産生増強方法。
7.前項5又は6に記載のHCV粒子の産生増強方法により産生されたHCVを不活化して作製する、HCVワクチンの作製方法。
8.前項7に記載の作製方法により作製された、HCVワクチン。
9.前項1〜4のいずれか1項に記載のHCV粒子形成促進剤の存在下で、抗HCV剤候補物質と共にHCV感染細胞を培養し、HCV粒子の形成を阻害する強さを評価する、抗HCV剤候補物質の評価方法。
10.以下の工程を含む、前項9に記載の抗HCV剤候補物質の評価方法:
1)前項1〜4のいずれか1項に記載のHCV粒子形成促進剤及び抗HCV剤候補物質を、HCV感染細胞に添加する工程;
2)HCV感染細胞を培養する培養工程;
3)培養されたHCV粒子の量を測定し、HCV粒子の形成を阻害する強さを評価する評価工程。That is, this invention consists of the following.
1. An HCV particle formation promoter containing a statin or a pharmaceutically acceptable salt thereof as an active ingredient.
2. 2. The HCV particle formation promoter according to item 1, wherein the statin is one or more statins selected from the group consisting of lovastatin, fluvastatin, simvastatin, atorvastatin, and pravastatin.
3. 3. The HCV particle formation promoter according to item 1 or 2, which contains a culture extract of a microorganism capable of producing a statin or a pharmaceutically acceptable salt thereof.
4). 4. The HCV particle formation promoter according to item 3 above, wherein the microorganism is an Aspergillus filamentous fungus.
5. A method for enhancing production of HCV particles, comprising culturing HCV-infected cells in the presence of the HCV particle formation promoter according to any one of items 1 to 4.
6). 6. The method for enhancing production of HCV particles according to item 5, wherein the HCV particle formation promoter according to any one of items 1 to 4 is added and cultured after the HCV protein is formed in the HCV-infected cells.
7). 7. A method for producing an HCV vaccine, which is produced by inactivating HCV produced by the method for enhancing production of HCV particles according to item 5 or 6.
8). An HCV vaccine produced by the production method according to item 7 above.
9. Anti-HCV, wherein HCV-infected cells are cultured with an anti-HCV agent candidate substance in the presence of the HCV particle formation promoter according to any one of items 1 to 4 above, and the strength to inhibit the formation of HCV particles is evaluated. Evaluation method for drug candidate substances.
10. The method for evaluating an anti-HCV agent candidate substance according to item 9 including the following steps:
1) A step of adding the HCV particle formation promoter and anti-HCV agent candidate substance according to any one of items 1 to 4 to HCV-infected cells;
2) a culture process for culturing HCV-infected cells;
3) An evaluation process for measuring the amount of cultured HCV particles and evaluating the strength to inhibit the formation of HCV particles.
本発明のHCV粒子形成促進剤を用いてHCV感染細胞を培養することにより、HCV粒子を形成促進させ、HCV粒子の産生効率を向上させることができた。本発明の方法より、HCV感染培養細胞でHCV粒子の産生を増加させることができると共に、抗HCV剤候補物質の評価を行うことができる。更に、本発明により得られたHCV粒子を用いてHCVワクチンを効率よく生産することができる。 By culturing HCV-infected cells using the HCV particle formation promoter of the present invention, it was possible to promote the formation of HCV particles and improve the production efficiency of HCV particles. According to the method of the present invention, production of HCV particles can be increased in cultured cells infected with HCV, and anti-HCV drug candidate substances can be evaluated. Furthermore, HCV vaccine can be efficiently produced using the HCV particles obtained by the present invention.
本発明は、スタチン又はその薬学的に許容しうる塩を有効成分とする、HCV粒子形成促進剤に関する。 The present invention relates to an HCV particle formation promoter comprising a statin or a pharmaceutically acceptable salt thereof as an active ingredient.
本明細書においてスタチンとは、コレステロールの生合成経路の一つメバロン酸経路の律速酵素であるHMG-CoA 還元酵素(3-Hydroxy-3-methylglutaryl coenzyme A reductase) の働きを阻害し肝臓でのコレステロールの生合成を抑制することにより高コレステロール血症の治療薬として使用されている薬物であり、ロバスタチン(Lovastatin)、シンバスタチン(Simvastatin)、フルバスタチンナトリウム(Fluvastatin sodium)、プラバスタチン(Pravastatin)、アトルバスタチンカルシウム(Atorvastatin Calcium)、ピタバスタチンカルシウム(Pitavastatin calcium)、ロスバスタチンカルシウム(Rosuvastatin calcium)等をいう。 In this specification, statins refer to cholesterol in the liver by inhibiting the action of HMG-CoA reductase (3-Hydroxy-3-methylglutaryl coenzyme A reductase), the rate-limiting enzyme of the mevalonate pathway, one of the biosynthetic pathways of cholesterol. It is a drug used as a treatment for hypercholesterolemia by inhibiting biosynthesis of lovastatin (Lovastatin), simvastatin, fluvastatin sodium, pravastatin (Pravastatin), atorvastatin calcium ( Atorvastatin Calcium), pitavastatin calcium, rosuvastatin calcium and the like.
ここにおいてロバスタチンとは、CAS番号75330-75-5、(1S,3R,7S,8S,8aR)-8-[2-((2R,4R)-4-ヒドロキシ-6-オキソテトラヒドロ-2H-ピラン-2-イル)エチル]-3,7-ジメチル-1,2,3,7,8,8a-ヘキサヒドロ-1-ナフチルを、シンバスタチンとは、CAS番号79902-63-9、(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-ヒドロキシ-6-オキソテトラヒドロピラン-2-イル]エチル]-3,7-ジメチル-1,2,3,7,8,8a-ヘキサヒドロ-1-ナフチルを、フルバスタチンナトリウムとは、CAS番号93957-55-2、(3R,5S,6E)-7-[3-(4-フルオロフェニル)-1-(1-メチルエチル)-1H-インドール-2-イル]-3,5-ジヒドロキシ-6-ヘプテン酸 ナトリウム、プラバスタチンとは、CAS番号81093-70-6、(1S,βR,δR,8aβ)-1,2,6,7,8,8a-ヘキサヒドロ-β,δ,6β-トリヒドロキシ-2β-メチル-8α-[(S)-2-メチル-1-オキソブトキシ]-1β-ナフタレンヘプタン酸を、アトルバスタチンカルシウムとは、CAS番号134523-03-8、(3R,5R)-7-[2-(4-フルオロフェニル)-5-イソプロピル-3-フェニル-4-[(フェニルアミノ)カルボニル]-1H-ピロール-1-イル]-3,5-ジヒドロキシヘプタン酸カルシウムを、ピタバスタチンカルシウムとは、CAS番号147511-69-1、(3R,5S,6E)-7-(2-シクロプロピル-4-(4-フルオルフェニル)キノリニ-3-イル)-3,5-ジヒドロキシヘプテ-6-ン酸を、ロスバスタチンカルシウムとは、CAS番号147098-20-2、ビス[(E)-7-[4-(4-フルオロフェニル)-6-イソプロピル-2-[メチル(メチルスルホニル) アミノ] ピリミジン-5-イル](3R,5S)-3,5-ジヒドロキシヘプテ-6-ン酸] カルシウムをいう。 Lovastatin here is CAS number 75330-75-5, (1S, 3R, 7S, 8S, 8aR) -8- [2-((2R, 4R) -4-hydroxy-6-oxotetrahydro-2H-pyran -2-yl) ethyl] -3,7-dimethyl-1,2,3,7,8,8a-hexahydro-1-naphthyl and simvastatin are CAS numbers 79902-63-9, (1S, 3R, 7S, 8S, 8aR) -8- [2-[(2R, 4R) -4-hydroxy-6-oxotetrahydropyran-2-yl] ethyl] -3,7-dimethyl-1,2,3,7, 8,8a-Hexahydro-1-naphthyl and fluvastatin sodium are CAS numbers 93957-55-2, (3R, 5S, 6E) -7- [3- (4-fluorophenyl) -1- (1- Methylethyl) -1H-indol-2-yl] -3,5-dihydroxy-6-heptenoate sodium, pravastatin is CAS No.81093-70-6, (1S, βR, δR, 8aβ) -1,2 , 6,7,8,8a-Hexahydro-β, δ, 6β-trihydroxy-2β-methyl-8α-[(S) -2-methyl-1-oxobutoxy] -1β-naphthaleneheptanoic acid, atorvastatin calcium And CAS number 13452 3-03-8, (3R, 5R) -7- [2- (4-Fluorophenyl) -5-isopropyl-3-phenyl-4-[(phenylamino) carbonyl] -1H-pyrrol-1-yl] -3,5-dihydroxyheptanoic acid and pitavastatin calcium are CAS numbers 147511-69-1, (3R, 5S, 6E) -7- (2-cyclopropyl-4- (4-fluorophenyl) quinolini -3-yl) -3,5-dihydroxyhepte-6-acid, rosuvastatin calcium, CAS number 147098-20-2, bis [(E) -7- [4- (4-fluorophenyl) -6-Isopropyl-2- [methyl (methylsulfonyl) amino] pyrimidin-5-yl] (3R, 5S) -3,5-dihydroxyhepte-6-acid] refers to calcium.
本発明のスタチンとして、上記より選択される1種又は複数種を選択して使用することができる。好ましくは、ロバスタチン、フルバスタチン、シンバスタチン、アトルバスタチン、及びプラバスタチンが挙げられ、より好ましくは、ロバスタチン、フルバスタチン、シンバスタチン、及びアトルバスタチンが挙げられる。本発明のHCV粒子形成促進剤に含まれる有効成分は、上述のスタチン又はその薬学的に許容しうる塩であってもよいし、それらの水和物であってもよい。 スタチン、その薬学的に許容しうる塩、及びそれらの水和物は、合成により作製してもよいし、微生物により産生させたものであってもよい。 As the statin of the present invention, one or more selected from the above can be selected and used. Preferred examples include lovastatin, fluvastatin, simvastatin, atorvastatin, and pravastatin, and more preferred examples include lovastatin, fluvastatin, simvastatin, and atorvastatin. The active ingredient contained in the HCV particle formation promoter of the present invention may be the above-mentioned statin or a pharmaceutically acceptable salt thereof, or a hydrate thereof. Statins, their pharmaceutically acceptable salts, and their hydrates may be made synthetically or produced by microorganisms.
本発明のHCV粒子形成促進剤は、合成により作製されたスタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を有効成分として含む組成物であってもよいし、スタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を産生しうる微生物の培養抽出物であってもよい。前記微生物の培養抽出物は、そのまま本発明のHCV粒子形成促進剤として用いることもできる。いずれの場合も、本発明のHCV粒子形成促進剤に含まれる有効成分は、スタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物である。スタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を産生しうる微生物の培養抽出物を、そのまま本発明のHCV粒子形成促進剤として用いることで、スタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を合成し、精製するよりもHCV粒子形成促進剤の製造を容易に行うことができ、費用も軽減化することができる。 The HCV particle formation promoter of the present invention may be a composition containing a synthetically produced statin, a pharmaceutically acceptable salt thereof, and / or a hydrate thereof as an active ingredient, a statin, It may be a cultivated extract of microorganisms capable of producing pharmaceutically acceptable salts and / or hydrates thereof. The microorganism culture extract can be used as it is as the HCV particle formation promoter of the present invention. In any case, the active ingredient contained in the HCV particle formation promoter of the present invention is a statin, a pharmaceutically acceptable salt thereof, and / or a hydrate thereof. By using a culture extract of a microorganism capable of producing a statin, a pharmaceutically acceptable salt, and / or a hydrate thereof as it is as an HCV particle formation promoter of the present invention, the statin, the pharmaceutically The production of the HCV particle formation accelerator can be facilitated and the cost can be reduced compared to the synthesis and purification of acceptable salts and / or hydrates thereof.
有効成分としてのスタチン、その薬学的に許容しうる塩、及びそれらの水和物は、合成によって作製することもできるし、生合成可能な微生物の培養抽出物から取得することもできる。スタチン、その薬学的に許容しうる塩、及びそれらの水和物を合成する方法は特に限定されず、自体公知の方法又は今後開発されるあらゆる合成方法を適用することができる。スタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を産生しうる微生物としては、特に限定されないが、例えばロバスタチンについてはAspergillus属糸状菌が挙げられ、特に好適にはAspergillus terreusが挙げられる(図1参照)。 Statins as active ingredients, pharmaceutically acceptable salts thereof, and hydrates thereof can be prepared synthetically or obtained from cultured extracts of biosynthesizable microorganisms. Methods for synthesizing statins, pharmaceutically acceptable salts thereof, and hydrates thereof are not particularly limited, and methods known per se or any synthetic method developed in the future can be applied. The microorganisms that can produce statins, pharmaceutically acceptable salts thereof, and / or hydrates thereof are not particularly limited, but for lovastatin, for example, Aspergillus filamentous fungi can be mentioned, and particularly preferably Aspergillus terreus (See FIG. 1).
本発明において薬学的に許容される塩とは、特に制限なく、当業者に公知の任意の塩、例えば、ナトリウム塩、カリウム塩、カルシウム塩などを挙げることが出来る。本発明のHCV粒子形成促進剤は、有効成分として上述のスタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物を含む他、他の化合物を含んでいてもよい。 In the present invention, the pharmaceutically acceptable salt includes, without particular limitation, any salt known to those skilled in the art, for example, sodium salt, potassium salt, calcium salt and the like. The HCV particle formation promoter of the present invention may contain other compounds in addition to the above-mentioned statin, its pharmaceutically acceptable salt, and / or hydrate thereof as an active ingredient.
本発明のHCV粒子形成促進剤の剤型は特に限定されず、有効成分としてのスタチン、その薬学的に許容しうる塩、及び/又はそれらの水和物をHCV感染細胞と共に培養可能であればよい。本発明のHCV粒子形成促進剤には、上記を考慮して、本発明の有効成分以外に当業者に公知の薬学的に許容され得る担体、賦形剤、結合剤、滑沢剤及び着色剤などを適宜含ませることができる。本発明のHCV粒子形成促進剤は、当業者に公知の任意の製剤調製方法で容易に調製することができる。 例えば、適当な担体の例としては、ラクトース、デンプン、ショ糖、グルコース、メチルセルロース、ステアリン酸マグネシウム、マンニトール、ソルビトール及びクロスカルメローズナトリウムなどを挙げることができる。或いは、適当な結合剤としては、デンプン、ゼラチン、又は、グルコース、無水ラクトース、自由流動ラクトース、ベータ-ラクトース及びトウモロコシ甘味料のような天然の糖、並びに、アラビアガム、グアーガム、トラガントもしくはアルギン酸ナトリウムのような天然及び合成のガム、カルボキシメチルセルロース、ポリエチレングリコール、及びロウなどがある。又、これらの剤形に使用される滑沢剤には、オレイン酸ナトリウム、ステアリン酸ナトリウム、ステアリン酸マグネシウム、安息香酸ナトリウム、及び塩化ナトリウムなどがある。 The dosage form of the HCV particle formation promoter of the present invention is not particularly limited as long as the statin as an active ingredient, a pharmaceutically acceptable salt thereof, and / or a hydrate thereof can be cultured with HCV-infected cells. Good. In consideration of the above, the HCV particle formation promoter of the present invention includes pharmaceutically acceptable carriers, excipients, binders, lubricants and colorants known to those skilled in the art in addition to the active ingredient of the present invention. Etc. can be included as appropriate. The HCV particle formation promoter of the present invention can be easily prepared by any formulation preparation method known to those skilled in the art. For example, examples of suitable carriers include lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and croscarmellose sodium. Alternatively, suitable binders include starch, gelatin or natural sugars such as glucose, anhydrous lactose, free flowing lactose, beta-lactose and corn sweeteners, and gum arabic, guar gum, tragacanth or sodium alginate. Natural and synthetic gums such as carboxymethylcellulose, polyethylene glycol, and waxes. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate and sodium chloride.
本発明は、上記HCV粒子形成促進剤と共にHCV感染細胞を培養することによる、HCV粒子の産生増強方法にも及ぶ。本発明のHCV粒子の産生に使用可能な細胞は、HCV許容性細胞であればよい。ここでHCV許容性細胞とは、HCVゲノムRNAの複製能及び/又はHCVが感染しうる細胞を意味する。HCV許容性細胞は、肝臓細胞又はリンパ球系細胞由来の細胞であるが、これらに限定されるものではない。肝臓細胞としては、具体的には初代肝臓細胞や、Huh7細胞、RCYM1RC細胞、5-15RC細胞、HepG2細胞、IMY-N9細胞、HeLa細胞、293細胞などが挙げられ、リンパ球系細胞としてはMolt4細胞や、HPB-Ma細胞、Daudi細胞などが挙げられるが、これらに限定されるものでは無い。好ましいHCV許容性細胞としては、Huh7細胞、RCYM1RC細胞、5-15RC細胞、HepG2細胞及びそれらの細胞から派生した株化細胞などが挙げられる。特に好ましくはHuh7細胞から派生した細胞であり、このような細胞としては、例えばHuh7.5細胞やHuh7.5.1細胞、Huh7-it細胞などが挙げられる。特に継代培養可能な細胞であれば好適である。さらに真核細胞であることが好ましく、ヒト細胞であることがより好ましい。これらの細胞は、市販のものを利用してもよいし、細胞寄託機関から入手して使用してもよい。任意の細胞(例えば癌細胞又は幹細胞)を株化した細胞を使用してもよい。Huh7細胞から派生した細胞株として、Huh7.5細胞(Blight KJ et al., J. Virol., 76:13001-13014, 2002)及びHuh7.5.1細胞(Zhong J et al., Proc. Natl. Acad. Sci. USA, 102:9294-9299, 2005)及びHuh7-it細胞(Yu L et al., J. Virol. Methods, 169:380-384, 2010)などが挙げられる。 The present invention also extends to a method for enhancing production of HCV particles by culturing HCV-infected cells together with the HCV particle formation promoter. The cells that can be used for production of the HCV particles of the present invention may be HCV-permissive cells. Here, the HCV-permissive cell means a cell capable of infecting HCV genomic RNA and / or HCV. HCV-permissive cells are cells derived from liver cells or lymphoid cells, but are not limited thereto. Specific examples of liver cells include primary liver cells, Huh7 cells, RCYM1RC cells, 5-15RC cells, HepG2 cells, IMY-N9 cells, HeLa cells, and 293 cells. Examples include, but are not limited to, cells, HPB-Ma cells, and Daudi cells. Preferred HCV-permissive cells include Huh7 cells, RCYM1RC cells, 5-15RC cells, HepG2 cells and cell lines derived from these cells. Particularly preferred are cells derived from Huh7 cells. Examples of such cells include Huh7.5 cells, Huh7.5.1 cells, and Huh7-it cells. Particularly, cells that can be subcultured are suitable. Further, eukaryotic cells are preferable, and human cells are more preferable. These cells may be commercially available or may be obtained from a cell depository. A cell in which an arbitrary cell (for example, a cancer cell or a stem cell) is established may be used. As cell lines derived from Huh7 cells, Huh7.5 cells (Blight KJ et al., J. Virol., 76: 13001-13014, 2002) and Huh7.5.1 cells (Zhong J et al., Proc. Natl. Acad) Sci. USA, 102: 9294-9299, 2005) and Huh7-it cells (Yu L et al., J. Virol. Methods, 169: 380-384, 2010).
本発明のHCV粒子形成促進剤を、上記より選択されるHCVが感染しているHCV許容性細胞に添加することによりHCV粒子を形成促進させ、HCV粒子を産生増強させることができる。具体的には、上記より選択されるいずれかの培養したHCV許容性細胞にHCVを接種し、HCVを細胞内に吸着させた後に、培養液に上記HCV粒子形成促進剤を、10〜200μg/ml、好ましくは20〜50μg/mlの濃度になるように添加し、24〜72時間、好ましくは36〜72時間、より好ましくは約48時間培養することでHCV粒子を形成促進させ、HCV粒子を産生増強させることができる。 By adding the HCV particle formation promoter of the present invention to HCV-permissive cells infected with HCV selected from the above, the formation of HCV particles can be promoted and the production of HCV particles can be enhanced. Specifically, after HCV is inoculated into any of the cultured HCV-permissive cells selected from the above and HCV is adsorbed inside the cells, the HCV particle formation promoter is added to the culture solution at 10 to 200 μg / ml, preferably 20 to 50 μg / ml, and cultivated for 24 to 72 hours, preferably 36 to 72 hours, more preferably about 48 hours to promote the formation of HCV particles. Production can be enhanced.
HCV感染細胞のHCV粒子産生能は、公知の任意のウイルス検出法を用いて確認することができる。例えば、HCV許容性細胞の培養上清を、ショ糖密度勾配により分画し、ウイルス粒子を検出することができる。HCVが感染し、HCVゲノムRNAが複製された細胞は、HCVタンパク質を発現する。従って、HCV感染細胞を培養し、HCVタンパク質を検出することができれば、その細胞はHCVゲノムRNAを複製しているものと推定することができる。さらに、HCVタンパク質の検出は、公知の任意のタンパク質検出法に従って行うことができる。具体的には、Kaito M et al., J. Gen. Virol., 75:l755-1760, 1994の方法により、検出することができる。ウイルス粒子産生能は、培養上清中の感染性ウイルス粒子の数を確認することで行うことができる。感染性ウイルス粒子を含む培養上清を非感染細胞に接種し、18〜48時間後、好ましくは約24時間後に細胞を固定して、HCVタンパク質に対する特異抗体を用いて免疫染色し、染色陽性細胞の数を計測することで、培養上清中の感染性ウイルス粒子の数を確認することができる。より具体的には、感染性ウイルス粒子を含む培養上清をEnzyme-linked Immunosorbent Assay(ELISA)法により抗HCV Coreタンパク質抗体を反応させ、検出することによって行うことができる。 The ability of HCV-infected cells to produce HCV particles can be confirmed using any known virus detection method. For example, the culture supernatant of HCV-permissive cells can be fractionated by a sucrose density gradient, and virus particles can be detected. Cells in which HCV is infected and HCV genomic RNA is replicated express HCV protein. Therefore, if HCV-infected cells can be cultured and HCV protein can be detected, it can be presumed that the cells are replicating HCV genomic RNA. Furthermore, HCV protein can be detected according to any known protein detection method. Specifically, it can be detected by the method of Kaito M et al., J. Gen. Virol., 75: l755-1760, 1994. The ability to produce virus particles can be performed by confirming the number of infectious virus particles in the culture supernatant. Inoculate non-infected cells with culture supernatant containing infectious virus particles, fix cells after 18-48 hours, preferably about 24 hours, immunostain with specific antibody against HCV protein, staining positive cells The number of infectious virus particles in the culture supernatant can be confirmed. More specifically, the culture supernatant containing infectious virus particles can be detected by reacting and detecting an anti-HCV Core protein antibody by an enzyme-linked immunosorbent assay (ELISA) method.
HCV感染細胞において複製されるHCV RNAの解析は、通常の分子生物学的方法で解析することができる。細胞からRNAを抽出する方法は、自体公知の方法によることができる。具体的には、ノーザンブロット法、リボヌクレアーゼプロテクションアッセイ法やRT-PCR法などを用いて、複製されたRNAの量又は配列を解析することができる。RNAの定量を行う場合はノーザンブロット法や定量RT-PCRで、RNAの配列を解析する場合はシークエンス解析法を用いることができる。 Analysis of HCV RNA replicated in HCV-infected cells can be analyzed by a normal molecular biological method. The method for extracting RNA from the cells can be a method known per se. Specifically, the amount or sequence of the replicated RNA can be analyzed using Northern blotting, ribonuclease protection assay, RT-PCR, or the like. When RNA is quantified, Northern blotting or quantitative RT-PCR can be used, and when RNA sequence is analyzed, sequence analysis can be used.
本発明の方法で産生されるHCV粒子は、HCV許容性細胞への感染能を有する。ここでHCV許容性細胞とは、HCVゲノムRNAの複製能及び/又はHCVが感染しうる細胞を意味する。HCV許容性細胞は、肝臓細胞又はリンパ球系細胞由来の細胞であるが、これらに限定されるものではない。肝臓細胞としては、具体的には初代肝臓細胞や、Huh7細胞、RCYM1RC細胞、5-15RC細胞、HepG2細胞、IMY-N9細胞、HeLa細胞、293細胞などが挙げられ、リンパ球系細胞としてはMolt4細胞や、HPB-Ma細胞、Daudi細胞などが挙げられるが、これらに限定されるものでは無い。好ましいHCV許容性細胞としては、Huh7細胞、RCYM1RC細胞、5-15RC細胞、HepG2細胞及びそれらの細胞から派生した株化細胞などが挙げられる。特に好ましくはHuh7細胞から派生した細胞であり、このような細胞としては、例えばHuh7.5細胞やHuh7.5.1細胞、Huh7-it細胞などが挙げられる。 HCV particles produced by the method of the present invention have the ability to infect HCV-permissive cells. Here, the HCV-permissive cell means a cell capable of infecting HCV genomic RNA and / or HCV. HCV-permissive cells are cells derived from liver cells or lymphoid cells, but are not limited thereto. Specific examples of liver cells include primary liver cells, Huh7 cells, RCYM1RC cells, 5-15RC cells, HepG2 cells, IMY-N9 cells, HeLa cells, and 293 cells. Examples include, but are not limited to, cells, HPB-Ma cells, and Daudi cells. Preferred HCV-permissive cells include Huh7 cells, RCYM1RC cells, 5-15RC cells, HepG2 cells and cell lines derived from these cells. Particularly preferred are cells derived from Huh7 cells. Examples of such cells include Huh7.5 cells, Huh7.5.1 cells, and Huh7-it cells.
感染性ウイルス粒子を含む培養上清など、上記で得られたHCV粒子を含むウイルス液から、HCV粒子を精製する方法は特に限定されず、自体公知の方法又は今後開発される方法を適用することができる。例えば、遠心及び/又はフィルターなどを用いて細胞及び細胞の残渣を除去し、限外濾過濃縮、クロマトグラフィー及び密度勾配遠心を任意の順番に組み合わせて、あるいは単独で精製することができる。 The method of purifying HCV particles from the virus solution containing HCV particles obtained above, such as a culture supernatant containing infectious virus particles, is not particularly limited, and a method known per se or a method developed in the future should be applied. Can do. For example, centrifugation and / or a filter can be used to remove cells and cell debris, and ultrafiltration concentration, chromatography and density gradient centrifugation can be combined in any order or purified alone.
本発明は、さらに本発明の方法で産生されるHCV粒子を抗原とするHCVワクチンの作製方法にも及ぶ。更に当該方法により作製されたHCVワクチンにも及ぶ。 The present invention further extends to a method for producing an HCV vaccine using HCV particles produced by the method of the present invention as an antigen. Furthermore, it extends to the HCV vaccine produced by this method.
本発明のHCVワクチンの作製に関しては、感染性が不活化されたHCV粒子を使用することが好ましい。感染性の不活化方法は臨床に使用可能な方法であればよく特に限定されず、自体公知の方法、今後開発される方法を採用することができる。例えばホルマリン、β−プロピオラクトン、グルタルジアルデヒド等の不活化剤を、例えば、本発明により作製されたHCV粒子浮遊液に添加混合し、HCV粒子と反応させることにより達成することができる(Appaiahgari MB et al., Vaccine, 22:3669-3675, 2004)。また、HCV粒子を紫外線で照射することで、感染性を失わせ、迅速に不活化することもできる。紫外線照射によれば、HCV粒子を構成するタンパク質などへの影響が少なく、不活化を行うことができる。不活化するための紫外線の線源としては一般に市販されている殺菌灯、特に15W殺菌灯を用いて行うことができるが、それらに限るものではない。 For the production of the HCV vaccine of the present invention, it is preferable to use HCV particles inactivated infectivity. The infectious inactivation method is not particularly limited as long as it is a clinically usable method, and a method known per se or a method developed in the future can be employed. For example, an inactivating agent such as formalin, β-propiolactone, and glutardialdehyde can be achieved by, for example, adding to and mixing with the HCV particle suspension prepared according to the present invention and reacting with the HCV particles (Appaiahgari MB et al., Vaccine, 22: 3669-3675, 2004). In addition, by irradiating HCV particles with ultraviolet rays, the infectivity can be lost and the HCV particles can be inactivated quickly. According to the ultraviolet irradiation, inactivation can be performed with little influence on proteins constituting HCV particles. The ultraviolet ray source for inactivation can be performed using a commercially available germicidal lamp, particularly a 15 W germicidal lamp, but is not limited thereto.
アジュバントは、ワクチン用アジュバントとして使用でき臨床に使用可能であればよく特に限定されないが、自体公知のアジュバント又は今後開発されるアジュバントを適用することができる。例えば、既にワクチン用アジュバントとして使用が認可されている水酸化アルミニウム(Alum)等が望ましいが、臨床に使用できるものならば良く、CpGオリゴヌクレオチド、2本鎖RNAが挙げられ、2本鎖RNA として、polyI:C、polyICLC又はpolyIpolyC12Uを挙げることができる。 The adjuvant is not particularly limited as long as it can be used as an adjuvant for vaccines and can be clinically used, and an adjuvant known per se or an adjuvant developed in the future can be applied. For example, aluminum hydroxide (Alum), which has already been approved for use as an adjuvant for vaccines, is desirable, but any substance that can be used clinically may be used, such as CpG oligonucleotides and double-stranded RNA. , PolyI: C, polyICLC or polyIpolyC12U.
本発明のHCV粒子形成促進剤は、さらに抗HCV剤候補物質の評価方法に利用することができる。ここにおいて、抗HCV剤は、細胞でのHCV粒子産生抑制作用や、細胞からのHCV粒子放出抑制作用を有する物質が挙げられる。抗HCV剤候補物質は、そのような作用が期待される物質であればよく、特に限定されないが、例えばタンパク質、ペプチドの他、低分子化合物などが挙げられる。 The HCV particle formation accelerator of the present invention can be further used in a method for evaluating anti-HCV agent candidate substances. Here, examples of the anti-HCV agent include a substance having an inhibitory action on HCV particle production in cells and an inhibitory action on HCV particle release from cells. The anti-HCV candidate substance is not particularly limited as long as it is a substance expected to have such an action, and examples thereof include proteins, peptides, and low molecular compounds.
抗HCV剤候補物質の評価方法としては、HCV感染細胞を本発明のHCV粒子形成促進剤と抗HCV剤候補物質と共に、あるいは抗HCV剤候補物質のみで36〜72時間培養し、培養HCV感染細胞からHCV粒子形成能を比較することで達成される。具体的には、以下の1)〜3)の工程を含む方法によることができる。
1)HCV感染細胞に、本発明のHCV粒子形成促進剤及び抗HCV剤候補物質を加える工程;
2)C型肝炎ウイルス感染細胞を培養する培養工程;
3)培養されたC型肝炎ウイルス粒子の量を測定し、C型肝炎ウイルス粒子の形成を阻害する強さを評価する評価工程。
ここで使用可能な細胞は、上述したHCV許容性細胞が挙げられる。As a method for evaluating an anti-HCV agent candidate substance, HCV-infected cells are cultured for 36 to 72 hours together with the HCV particle formation promoter of the present invention and the anti-HCV agent candidate substance or only with the anti-HCV agent candidate substance, and cultured HCV-infected cells This is achieved by comparing the HCV particle formation ability. Specifically, it can be based on a method including the following steps 1) to 3).
1) A step of adding the HCV particle formation promoter and anti-HCV agent candidate substance of the present invention to HCV-infected cells;
2) a culture process for culturing hepatitis C virus-infected cells;
3) An evaluation step of measuring the amount of cultured hepatitis C virus particles and evaluating the strength to inhibit the formation of hepatitis C virus particles.
Examples of the cells that can be used here include the above-described HCV-permissive cells.
本発明の理解を深めるために、本発明の内容を実施例により具体的に説明するが、本発明はこれら実施例に限定されるものではないことは明らかである。 In order to deepen the understanding of the present invention, the contents of the present invention will be specifically described with reference to examples. However, it is obvious that the present invention is not limited to these examples.
(実施例1)HCV粒子形成促進剤
本実施例では、本発明のHCV粒子形成促進剤として、Aspergillus terreus培養抽出物(B13)を原料とする溶液を使用した。本実施例のHCV粒子形成促進剤は、図1に示す方法で作製した。以下の実施例では、図1に示す粗製ロバスタチン溶液「B15」又は精製ロバスタチン溶液「B15.4.1」をHCV粒子形成促進剤として使用した。B15.4.1の組成を核磁気共鳴(NMR)法及び液体クロマトグラフィー・マススペクトロメトリー(LC/MS)により調べた結果、ロバスタチンがほぼ純品で得られたことが確認された。ロバスタチン量は、乾燥させた精製標品(B15.4.1)を精密化学天秤にて秤量し、計測した。(Example 1) HCV particle formation promoter In this example, a solution using Aspergillus terreus culture extract (B13) as a raw material was used as the HCV particle formation promoter of the present invention. The HCV particle formation accelerator of this example was produced by the method shown in FIG. In the following Examples, the crude lovastatin solution “B15” or the purified lovastatin solution “B15.4.1” shown in FIG. 1 was used as an HCV particle formation accelerator. As a result of examining the composition of B15.4.1 by nuclear magnetic resonance (NMR) method and liquid chromatography / mass spectrometry (LC / MS), it was confirmed that lovastatin was obtained in a substantially pure product. The amount of lovastatin was measured by weighing the dried purified sample (B15.4.1) with a fine chemical balance.
(実施例2)培養細胞を処理したときのHCV感染価について
本実施例では、HCV粒子形成促進剤をHuh7-it細胞に添加したときのHCV感染価に及ぼす影響を確認した。(Example 2) HCV infectivity when cultured cells were treated In this example, the effect on the HCV infectivity when an HCV particle formation promoter was added to Huh7-it cells was confirmed.
1)HCV粒子形成促進剤
実施例では、実施例1の精製ロバスタチン溶液「B15.4.1」(図1)を「HCV粒子形成促進剤」として使用した。以下ではHCV粒子形成促進剤に培養液を加えて、ウイルス接種時及び培養時のロバスタチン濃度が1.25〜20μg/mlとなるように調整し、使用した。1) HCV particle formation accelerator In the examples, the purified lovastatin solution “B15.4.1” (FIG. 1) of Example 1 was used as an “HCV particle formation accelerator”. In the following, a culture solution was added to the HCV particle formation promoter so that the lovastatin concentration at the time of virus inoculation and culture was adjusted to 1.25 to 20 μg / ml.
2)培養細胞
本実施例では、培養細胞として、Huh7細胞由来のHCV高感受性株Huh7-it細胞を用いた。培地は、10%牛胎児血清・非必須アミノ酸・ペニシリン・ストレプトマイシン添加Dulbecco's modified Eagle's培地を使用し、48ウェル培養プレートを用いて培養した。2) Cultured cells In this Example, Huh7 cell-derived HCV hypersensitive strain Huh7-it cells were used as cultured cells. As the medium, Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, non-essential amino acids, penicillin, and streptomycin was used and cultured in a 48-well culture plate.
3)HCVストック液
本発明のHCV粒子形成促進のために使用するHCVストック液は、以下の方法で調製した。HCV株はJFH1株(ゲノム配列:GenBank accession number AB047639)を使用した。HCV JFH1株フルゲノムと同一配列のRNAを、Huh7-it細胞にエレクトロポレーション法(van den Hoff MJ et al., Nucleic Acids Res., 20:2902, 1992)にて導入(トランスフェクション)し、導入72時間後の培養上清を回収した。回収した培養上清は、0.45μmのフィルター(Millipore社)に通して夾雑物を除いた後、別のHuh7-it細胞に添加し、72時間後のHCV感染細胞数をフォーカス法により計測することにより感染力価を算出し、5.4×104 感染単位/mlに調整したものをHCVストック液とした。HCVストック液調製のための感染力価は、免疫染色法により行った。1次抗体として抗HCV-Core(クローンCP14)モノクローナル抗体を用い、標識抗体としてHRP標識ヤギ抗マウス抗体を用いた。コニカイムノステインHRP-1000(コニカミノルタ社)を加え、青色に染色したウイルス抗原陽性細胞集団(免疫フォーカス;フォーカスとも呼ぶ)の数を顕微鏡下で測定し、感染力価を算出した。3) HCV stock solution The HCV stock solution used for promoting the HCV particle formation of the present invention was prepared by the following method. As the HCV strain, the JFH1 strain (genome sequence: GenBank accession number AB047639) was used. RNA with the same sequence as the full genome of HCV JFH1 strain was introduced (transfected) into Huh7-it cells by electroporation (van den Hoff MJ et al., Nucleic Acids Res., 20: 2902, 1992). The culture supernatant after 72 hours was collected. Pass the collected culture supernatant through a 0.45 μm filter (Millipore) to remove impurities, add to another Huh7-it cell, and count the number of HCV-infected cells 72 hours later using the focus method. The infectious titer was calculated by the above and adjusted to 5.4 × 10 4 infectious units / ml was used as the HCV stock solution. The infectious titer for preparing the HCV stock solution was determined by immunostaining. An anti-HCV-Core (clone CP14) monoclonal antibody was used as the primary antibody, and an HRP-labeled goat anti-mouse antibody was used as the labeled antibody. Konica Immunostain HRP-1000 (Konica Minolta) was added, and the number of virus antigen-positive cell populations (immune focus; also called focus) stained in blue was measured under a microscope to calculate the infectious titer.
4)HCV感染方法
上記3)で調製したHCVストック液(5.4×104 感染単位/ml)とHCV粒子形成促進剤を混合したものを培養細胞に接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、同様にHCV粒子形成促進剤を加えて48時間培養した(ロバスタチン濃度:1.25〜20μg/ml)。ウイルスは、多重感染価(multiplicity of infection; moi)が0.1となるように培養細胞に接種した。4) HCV infection method After inoculating cultured cells with a mixture of the HCV stock solution (5.4 × 10 4 infection units / ml) prepared in 3) above and an HCV particle formation promoter, and adsorbing them at 37 ° C. for 2 hours After removing the virus solution and washing with the culture solution three times, HCV particle formation promoter was added in the same manner and cultured for 48 hours (lovastatin concentration: 1.25 to 20 μg / ml). The virus was inoculated into the cultured cells so that the multiplicity of infection (moi) was 0.1.
5)ウイルス感染価測定方法
ウイルス産生能を確認するために、上記感染細胞培養液を10,000 rpmで3分間遠心し、その遠心上清を試料液とした。上記試料液をHCV非感染Huh7-it細胞に接種し、24時間後に細胞を固定して、一次抗体としてHCVタンパク質に強く反応することが予め確認された患者血清、及び二次抗体としてAlexa488標識ヤギ抗ヒトIgG抗体(Molecular Probe社)を用いて免疫染色し、染色陽性細胞の数を計測し、HCV感染価を測定した。5) Method for measuring virus infectivity value In order to confirm the virus production ability, the infected cell culture solution was centrifuged at 10,000 rpm for 3 minutes, and the centrifuged supernatant was used as a sample solution. The above sample solution is inoculated into HCV-uninfected Huh7-it cells, and after 24 hours, the cells are fixed, patient serum that has been confirmed in advance to react strongly with HCV protein as a primary antibody, and Alexa488-labeled goat as a secondary antibody. Immunostaining was performed using an anti-human IgG antibody (Molecular Probe), the number of staining positive cells was counted, and the HCV infectivity titer was measured.
6)ウイルス感染価測定結果
上記の結果、5〜20μg/mlのロバスタチンが含まれるように調整された精製ロバスタチン溶液「B15.4.1」存在下で培養した場合に、ロバスタチン濃度依存的に各試料液について高いHCV感染価が認められた(図2)。6) Virus infectivity titer measurement results When cultured in the presence of the purified lovastatin solution “B15.4.1” adjusted to contain 5 to 20 μg / ml of lovastatin, each sample solution depends on the lovastatin concentration. A high HCV infection titer was observed for (Fig. 2).
(実施例3)培養細胞へのHCV粒子形成促進剤の添加時期の検討(1)
本実施例では、HCV粒子形成促進剤をHCV吸着中2時間及び/又は感染後48時間にHuh7-it細胞に添加したときのHCV感染価に及ぼす影響を確認した。(Example 3) Examination of timing of addition of HCV particle formation promoter to cultured cells (1)
In this example, the effect on the HCV infectivity titer when HCV particle formation promoter was added to Huh7-it cells for 2 hours during HCV adsorption and / or 48 hours after infection was confirmed.
1)HCV粒子形成促進剤
本実施例では、実施例1の粗製ロバスタチン溶液「B15」(図1)を「HCV粒子形成促進剤」として使用した。以下では、HCV粒子形成促進剤に培養液を加えて、ウイルス接種時及び培養時のロバスタチン濃度が1〜50μg/mlとなるように調整し、使用した。
2)培養細胞
本実施例では、実施例2と同手法でHuh7-it細胞を培養し、使用した。
3)HCV粒子形成促進剤の添加時期
本実施例ではHCVを実施例2と同様にmoi=0.1となるように培養細胞に接種し、HCV粒子形成促進剤は以下のi)〜iii)の時期に添加した。
i)吸着中+吸着後
培養細胞に、HCVとHCV粒子形成促進剤を混合したものを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、ロバスタチンを含むHCV粒子形成促進剤を加えて46時間培養した。
ii)吸着後のみ
培養細胞にHCVを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤添加培養液を加えて46時間培養した。
iii))吸着中のみ
培養細胞にHCVとHCV粒子形成促進剤を混合したものを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤を含まない培養液を加えて46時間培養した。
4)ウイルス感染価測定方法
ウイルス産生能を確認するための試料は実施例2と同手法にて調製し、実施例2と同手法でウイルス感染価を測定した。
5)ウイルス感染価測定結果
上記の結果、HCV吸着中にHCV粒子形成促進剤が存在するか否かに関わらず、HCV吸着以降にHCV粒子形成促進剤を添加して培養した場合に、5〜20μg/mlのロバスタチン存在下で濃度依存的に各試料液について高いウイルス感染価が認められた(図3)。1) HCV particle formation accelerator In this example, the crude lovastatin solution “B15” (FIG. 1) of Example 1 was used as an “HCV particle formation accelerator”. In the following, a culture solution was added to the HCV particle formation promoter to adjust the lovastatin concentration at the time of virus inoculation and culture to 1 to 50 μg / ml.
2) Cultured cells In this example, Huh7-it cells were cultured and used in the same manner as in Example 2.
3) Addition timing of HCV particle formation accelerator In this example, HCV was inoculated into cultured cells so that moi = 0.1 as in Example 2, and the HCV particle formation accelerator was the following i) to iii) Added to.
i) During adsorption + After adsorption After inoculating the cultured cells with a mixture of HCV and HCV particle formation promoter, adsorbed at 37 ° C for 2 hours, removed the virus solution, washed 3 times with the culture solution, and then lovastatin HCV particle formation promoter containing was added and cultured for 46 hours.
ii) Only after adsorption After inoculating the cultured cells with HCV and adsorbing at 37 ° C for 2 hours, removing the virus solution, washing 3 times with the culture solution, adding the culture solution with HCV particle formation promoter added, and culturing for 46 hours did.
iii)) Only during adsorption After inoculating cultured cells with a mixture of HCV and HCV particle formation promoter, adsorbed at 37 ° C for 2 hours, removed the virus solution, washed 3 times with the culture solution, and then formed HCV particles A culture solution containing no promoter was added and cultured for 46 hours.
4) Method for measuring virus infectivity titer A sample for confirming virus-producing ability was prepared in the same manner as in Example 2, and the virus infectivity titer was measured in the same manner as in Example 2.
5) Virus infectivity titer measurement results As a result of the above, when HCV particle formation promoter is added after HCV adsorption and cultured regardless of whether HCV particle formation promoter is present during HCV adsorption, In the presence of 20 μg / ml lovastatin, a high virus infectivity value was observed for each sample solution in a concentration-dependent manner (FIG. 3).
(実施例4)培養細胞へのHCV粒子形成促進剤の添加時期の検討(2)
本実施例では、HCV粒子形成促進剤としてロバスタチンを、HCV吸着中2時間又は感染後48時間にHuh7-it細胞に添加したときのHCV感染価に及ぼす影響を確認した。(Example 4) Examination of timing of addition of HCV particle formation promoter to cultured cells (2)
In this example, the effect of lovastatin as an HCV particle formation promoter on HCV infectivity when added to Huh7-it cells for 2 hours during HCV adsorption or 48 hours after infection was confirmed.
1)HCV粒子形成促進剤
本実施例では、実施例1の精製ロバスタチン溶液「B15.4.1」(図1)を「HCV粒子形成促進剤」として使用した。以下では、HCV粒子形成促進剤に培養液を加えて、ウイルス接種時及び培養時のロバスタチン濃度が20μg/mlとなるように調整し、使用した。
2)培養細胞
本実施例では、実施例2と同手法でHuh7-it細胞を培養し、使用した。
3)HCV粒子形成促進剤の添加時期
本実施例ではHCVを実施例2と同様にmoi=0.1となるように培養細胞に接種し、HCV粒子形成促進剤は以下のi)〜iii)の時期に添加した。
i)吸着中+吸着後
培養細胞にHCVとHCV粒子形成促進剤を混合したものを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤を添加した培養液を加えて46時間培養した。
ii)吸着中のみ
培養細胞にHCVとHCV粒子形成促進剤を混合したものを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤を含まない培地を加えて46時間培養した。
iii)吸着後のみ
培養細胞にHCVを接種して37℃で2時間吸着させた後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤を添加した培養液を加えて46時間培養した。
4)ウイルス感染価測定方法
ウイルス産生能を確認するための試料は実施例2と同手法にて調製し、実施例2と同手法でウイルス感染価を測定した。
5)ウイルス感染価測定結果
上記の結果、HCV吸着中にHCV粒子形成促進剤が存在するか否かに関わらず、HCV吸着以降にHCV粒子形成促進剤を添加して培養した場合に、高いウイルス感染価が認められた(図4)。1) HCV particle formation accelerator In this example, the purified lovastatin solution “B15.4.1” (FIG. 1) of Example 1 was used as an “HCV particle formation accelerator”. In the following, a culture solution was added to the HCV particle formation promoter to adjust the lovastatin concentration at the time of virus inoculation and culture to 20 μg / ml.
2) Cultured cells In this example, Huh7-it cells were cultured and used in the same manner as in Example 2.
3) Addition timing of HCV particle formation accelerator In this example, HCV was inoculated into cultured cells so that moi = 0.1 as in Example 2, and the HCV particle formation accelerator was the following i) to iii) Added to.
i) During adsorption + After adsorption After inoculating the cultured cells with a mixture of HCV and HCV particle formation promoter, adsorbed at 37 ° C for 2 hours, removed the virus solution, washed 3 times with the culture solution, and then HCV particles The culture solution to which the formation promoter was added was added and cultured for 46 hours.
ii) Only during adsorption After inoculating the cultured cells with a mixture of HCV and HCV particle formation promoter, adsorbed at 37 ° C for 2 hours, removed the virus solution, washed 3 times with the culture solution, and then promoted HCV particle formation. A medium containing no agent was added and cultured for 46 hours.
iii) Only after adsorption After inoculating HCV on cultured cells and adsorbing at 37 ° C. for 2 hours, the virus solution was removed, washed 3 times with the culture solution, and then added with a culture solution to which an HCV particle formation promoter was added. Incubate for hours.
4) Method for measuring virus infectivity titer A sample for confirming virus-producing ability was prepared in the same manner as in Example 2, and the virus infectivity titer was measured in the same manner as in Example 2.
5) Virus infectivity titer measurement results As a result of the above, regardless of whether or not an HCV particle formation accelerator is present during HCV adsorption, a high virus is observed when HCV particle formation accelerator is added and cultured after HCV adsorption. Infectious titer was observed (Figure 4).
(実施例5)培養細胞内外でのHCV粒子形成確認
本実施例では、培養細胞は実施例2と同手法により準備し、HCV吸着中から感染後48時間にわたってHCV粒子形成促進剤を、Huh7-it細胞に添加したときの、細胞内外でのHCV感染価に及ぼす影響を確認した。本実施例では、HCV粒子形成促進剤として、実施例1の精製ロバスタチン溶液「B15.4.1」(図1)を使用した。以下では、HCV粒子形成促進剤に培養液を加えて、ウイルス接種時及び培養時のロバスタチン濃度が20μg/mlとなるように調整し、使用した。HCV粒子形成促進剤のかわりにジメチルスルホキシド (DMSO)を含む系をコントロールとした。(Example 5) Confirmation of HCV particle formation inside and outside cultured cells In this example, cultured cells were prepared by the same method as in Example 2, and an HCV particle formation promoter was added for 48 hours after infection from during HCV adsorption. The effect on the HCV infectivity inside and outside the cell when added to it cells was confirmed. In this example, the purified lovastatin solution “B15.4.1” (FIG. 1) of Example 1 was used as an HCV particle formation accelerator. In the following, a culture solution was added to the HCV particle formation promoter to adjust the lovastatin concentration at the time of virus inoculation and culture to 20 μg / ml. A system containing dimethyl sulfoxide (DMSO) instead of the HCV particle formation accelerator was used as a control.
細胞外のHCV感染価を測定するための試料は、実施例2と同手法によった。細胞内のHCV感染価を測定するための試料は、凍結融解法で調製した。まず、感染細胞をPBSで洗浄後、トリプシン/EDTA処理により浮遊させ、遠心して細胞ペレットを回収した。それをPBSで1回洗浄し、0.5 mlの培養液に懸濁して-80℃で3回凍結融解を繰り返した後、12,000rpmで5分間遠心して得られた上清を測定用試料とした。各試料についてのウイルス力価測定は、実施例2と同手法により行った。 The sample for measuring the extracellular HCV infectivity titer was the same as in Example 2. Samples for measuring intracellular HCV infectivity were prepared by the freeze-thaw method. First, the infected cells were washed with PBS, suspended by trypsin / EDTA treatment, and centrifuged to collect the cell pellet. It was washed once with PBS, suspended in 0.5 ml culture medium, freeze-thawed three times at -80 ° C., and then centrifuged at 12,000 rpm for 5 minutes to obtain a supernatant as a measurement sample. The virus titer measurement for each sample was performed in the same manner as in Example 2.
上記の結果、細胞内ではHCV粒子産生はコントロールとほとんど差を認めなかったが、細胞外ではHCV粒子形成促進剤で処理した系のほうが20倍〜30倍と有意に高いHCV粒子産生能が確認された(図5)。 As a result of the above, HCV particle production in cells was almost the same as that in the control, but the cells treated with HCV particle formation accelerators were found to have significantly higher HCV particle production ability of 20 to 30 times extracellularly. (FIG. 5).
(実施例6)培養細胞内でのHCV RNAコピー数の確認
本実施例では、培養細胞は実施例2と同手法により準備し、HCV吸着中から感染後48時間にわたってHCV粒子形成促進剤を、Huh7-it細胞に添加したときの、HCV感染後1、2、3、4日目での、培養細胞内でのHCV RNAコピー数に及ぼす影響を確認した。本実施例では、HCV粒子形成促進剤として、実施例1の精製ロバスタチン溶液「B15.4.1」(図1)を使用した。以下では、HCV粒子形成促進剤に培養液を加えて、ウイルス接種時及び培養時のロバスタチン濃度が20μg/mlとなるように調整し、使用した。HCV粒子形成促進剤のかわりにDMSOを含む系をコントロールとした。(Example 6) Confirmation of the number of HCV RNA copies in cultured cells In this example, cultured cells were prepared by the same method as in Example 2, and HCV particle formation promoters were added for 48 hours after infection from during HCV adsorption. The effect of adding to Huh7-it cells on the number of HCV RNA copies in cultured cells on days 1, 2, 3, and 4 after HCV infection was confirmed. In this example, the purified lovastatin solution “B15.4.1” (FIG. 1) of Example 1 was used as an HCV particle formation accelerator. In the following, a culture solution was added to the HCV particle formation promoter to adjust the lovastatin concentration at the time of virus inoculation and culture to 20 μg / ml. A system containing DMSO instead of the HCV particle formation accelerator was used as a control.
細胞内のHCV RNAコピーを計測するための試料は、実施例5と同手法で調製した。感染細胞からTrizol(R)/Trizol(R)-LS(Invitrogen社)を用いて全RNAを抽出した。全RNAから、ReverTra Ace(R) qPCR-Kit (Toyobo)を用いて、37℃15分間逆転写反応、98℃5分間で酵素失活反応を行い、cDNAを合成した。HCV RNAコピー数の測定は、LightCycler(R) 480 リアルタイムPCRシステムを用いた定量RT-PCR法により行った。HCV特異的プライマーとして配列番号1及び2に示す塩基配列からなる、NS3部位へのプライマーを用い、PCR反応溶液は10μl 2×SYBR(R) Premix ExTaq(Takara)、0.4μl 各プライマー(10 pmol/μl)、2μl cDNAテンプレート、7.2μl 滅菌蒸留水を用いた。PCR条件は95℃、10秒の熱変性後、95℃10秒、60℃20秒のサイクルを40回繰り返した。
(配列番号1)5'-CTTTGACTCCGTGATCGACT-3'
(配列番号2)5'-CCCTGTCTTCCTCTACCTG-3 'A sample for measuring intracellular HCV RNA copy was prepared in the same manner as in Example 5. From infected cells using Trizol (R) / Trizol (R ) -LS (Invitrogen Co.) to extract total RNA. From total RNA, using the ReverTra Ace (R) qPCR-Kit (Toyobo), 37 ℃ 15 min reverse transcription reaction, the enzyme deactivation reaction at 98 ° C. 5 minutes to synthesize cDNA. Measurements of the number of HCV RNA copies was carried out by LightCycler (R) quantitative RT-PCR method using the 480 real-time PCR system. A primer for the NS3 site consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 was used as the HCV-specific primer. The PCR reaction solution was 10 μl 2 × SYBR (R) Premix ExTaq (Takara), 0.4 μl of each primer (10 pmol / μl), 2 μl cDNA template, and 7.2 μl sterile distilled water. PCR conditions were as follows: heat denaturation at 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 10 seconds and 60 ° C. for 20 seconds.
(SEQ ID NO: 1) 5'-CTTTGACTCCGTGATCGACT-3 '
(SEQ ID NO: 2) 5'-CCCTGTCTTCCTCTACCTG-3 '
上記の実験結果より、細胞内HCV RNA複製量はHCV粒子形成促進剤の有無によって影響を受けないことがわかった(図6)。また、免疫染色により、細胞内HCVタンパク質(抗原)合成量もロバスタチンの有無によって影響を受けないことがわかった(図7)。これらの実験結果より、ロバスタチンのHCV産生亢進作用は、HCV RNA複製やHCVタンパク質合成までの段階ではなく、それ以降のHCV粒子形成あるいは放出の段階で作用しているものと推測された。 From the above experimental results, it was found that the amount of intracellular HCV RNA replication was not affected by the presence or absence of an HCV particle formation promoter (FIG. 6). In addition, immunostaining revealed that the amount of intracellular HCV protein (antigen) synthesis was not affected by the presence or absence of lovastatin (FIG. 7). From these experimental results, it was speculated that the HCV production-enhancing effect of lovastatin is not at the stage until HCV RNA replication or HCV protein synthesis, but at the subsequent stage of HCV particle formation or release.
(実施例7)各種スタチンのHCV粒子産生促進効果
本実施例では、市販されている各種スタチンについて、HCV粒子産生促進効果を観察した。(Example 7) HCV particle production promoting effect of various statins In this example, HCV particle production promoting effect was observed for various commercially available statins.
1)HCV粒子形成促進剤
本実施例では、HCV粒子形成促進剤として市販のスタチン製剤を使用した。ロバスタチン(メビノリン:シグマアルドリッチ社製;CAS番号75330-75-5)、フルバスタチンナトリウム(和光純薬工業株式会社製;CAS番号93957-55-2)、シンバスタチン(シグマアルドリッチ社製;CAS番号79902-63-9)、アトルバスタチンカルシウム三水和物(和光純薬工業株式会社製;CAS番号134523-03-8)及びプラバスタチンナトリウム塩水和物(和光純薬工業株式会社製;CAS番号81131-70-6)を用いた。各スタチンは、培養時の濃度が表1に示す濃度になるように培養液で希釈して調整した。コントロールとしてHCV粒子形成促進剤のかわりにDMSO(1μg/ml)を用いた。
2)培養細胞
本実施例では、実施例2と同手法でHuh7-it細胞を培養し、使用した。
3)HCV粒子形成促進剤の添加時期
本実施例ではHCVをmoi=0.1となるように培養細胞に接種して37℃で2時間吸着後、ウイルス液を除き、培養液で3回洗浄後、HCV粒子形成促進剤を添加した培養液を加えて46時間培養した。
4)ウイルス感染価測定方法
ウイルス産生能を確認するための試料は実施例2と同手法にて調製し、実施例2と同手法でウイルス感染価を測定した。
5)ウイルス感染価測定結果
上記の結果を表1に示した。その結果、各スタチンについてHCV粒子形成促進剤効果が認められた。1) HCV particle formation accelerator In this example, a commercially available statin preparation was used as an HCV particle formation accelerator. Lovastatin (mevinolin: Sigma-Aldrich; CAS No. 75330-75-5), fluvastatin sodium (Wako Pure Chemical Industries, Ltd .; CAS No. 93957-55-2), simvastatin (Sigma-Aldrich; CAS No. 79902- 63-9), atorvastatin calcium trihydrate (Wako Pure Chemical Industries, Ltd .; CAS No. 134523-03-8) and pravastatin sodium salt hydrate (Wako Pure Chemical Industries, Ltd .; CAS No. 81131-70-6) ) Was used. Each statin was adjusted by diluting with a culture solution so that the concentration at the time of culture became the concentration shown in Table 1. As a control, DMSO (1 μg / ml) was used instead of the HCV particle formation accelerator.
2) Cultured cells In this example, Huh7-it cells were cultured and used in the same manner as in Example 2.
3) Addition timing of HCV particle formation promoter In this example, HCV was inoculated into the cultured cells so that moi = 0.1, adsorbed at 37 ° C. for 2 hours, the virus solution was removed, washed 3 times with the culture solution, The culture solution to which the HCV particle formation accelerator was added was added and cultured for 46 hours.
4) Method for measuring virus infectivity titer A sample for confirming virus-producing ability was prepared in the same manner as in Example 2, and the virus infectivity titer was measured in the same manner as in Example 2.
5) Virus infectivity titer measurement results The results are shown in Table 1. As a result, HCV particle formation promoter effect was recognized for each statin.
以上詳述したように、本発明のHCV粒子形成促進剤を添加してHCV感染細胞を培養することにより、細胞培養液にHCV粒子の産生効率を10倍以上向上させることが期待できる。これにより効果的にHCVワクチンを効率よく生産することができる。さらに、HCV感染細胞を本発明のHCV粒子形成促進剤及び抗HCV剤候補物質と共に培養し、細胞培養液中のウイルス力価及び/又はHCV RNAコピー数の計測を行うことで、抗HCV剤の評価を行うことができる。また、更に、本発明により得られたHCV粒子を用いてHCVワクチンを効率よく生産することができる。 As described above in detail, by adding the HCV particle formation promoter of the present invention and culturing HCV-infected cells, it can be expected to improve the production efficiency of HCV particles in the cell culture medium by 10 times or more. Thereby, an HCV vaccine can be produced efficiently and efficiently. Furthermore, by culturing HCV-infected cells with the HCV particle formation promoter and anti-HCV agent candidate substance of the present invention and measuring the virus titer and / or HCV RNA copy number in the cell culture medium, the anti-HCV agent Evaluation can be made. Furthermore, an HCV vaccine can be efficiently produced using the HCV particles obtained according to the present invention.
Claims (10)
1)請求項1〜4のいずれか1項に記載のC型肝炎ウイルス粒子形成促進剤及び抗C型肝炎ウイルス剤候補物質を、C型肝炎ウイルス感染細胞に添加する工程;
2)C型肝炎ウイルス感染細胞を培養する培養工程;
3)培養されたC型肝炎ウイルス粒子の量を測定し、C型肝炎ウイルス粒子の形成を阻害する強さを評価する評価工程。The method for evaluating an anti-hepatitis C virus candidate substance according to claim 9, comprising the following steps:
1) A step of adding the hepatitis C virus particle formation promoter and anti-hepatitis C virus agent candidate substance according to any one of claims 1 to 4 to hepatitis C virus-infected cells;
2) a culture process for culturing hepatitis C virus-infected cells;
3) An evaluation step of measuring the amount of cultured hepatitis C virus particles and evaluating the strength to inhibit the formation of hepatitis C virus particles.
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